Neuronal differentiation is a consequence of extrinsic and intrinsic regulatory cascades that ultimately acts to regulate gene expression and leads to mature neurons that express a limited set of genes encoding proteins that perform very specific functions. Part of this repertoire of genes are those that encode proteins required for neuronal signaling, including neurotransmitter biosynthetic enzymes and their cognate neurotransmitter receptors. Signaling through nicotinic acetylcholine (nACh) receptors mediates a variety of behaviors ranging from muscle contraction to memory formation. Not surprisingly, compromised signaling through these receptors is implicated in a number of neurological disorders. Despite these critical functions, relatively little is known regarding the molecular details underlying nACh receptor expression. The long-term goal of this project is to understand the molecular underpinnings of neuronal nACh receptor gene expression with a particular emphasis on the genomically clustered beta4/alpha3/alpha5subunit genes. Using in vitro approaches, we previously identified a cell-type-specific transcriptional regulatory region of the beta4 subunit gene. Our recent studies indicate that this region directs cell type-specific gene expression in vivo. In addition, we generated data suggesting the presence of a transcriptional represser in an intron of one of the clustered receptor genes. Using a multifacted approach, we will address the following Aims in this proposal: 1) to fully characterize, in vivo, the transcriptional activity of the beta4 regulatory region, 2) to determine the physiological importance of two transcriptional control elements within the beta4 region, 3) to test the hypothesis that, in the context of native chromatin, interactions between previously identified regulatory factors control beta4 gene expression and 4) to characterize the potential represser. These studies will provide valuable information regarding regulation of the neuronal nACh receptor gene family as well as contribute significantly to our understanding of neuronal development and differentiation. Our research is aimed at understanding, in molecular detail, how the expression of genes encoding proteins involved in nervous system function is regulated. By elucidating the mechanisms controlling when and where these genes are expressed, we will be better positioned to develop therapeutic interventions for a variety of neurological disorders.